Description of method of sampling used in microbiology

1. Conventional methods and
sampling of food

2. Introduction
• Food is the basic need of the human
• Commercialization of food
• Spoilage and outbreaks
• Emergence of food safety and bio-security
• Microbial analysis and food analysis

3. Methods used
• Microbial evaluation of food and food ingredients and environments
• Broadly grouped into Quantitative and Qualitative methods
• Quantitative methods – growth condition
• Qualitative methods – for identification

4. Standard and Recommended methods
• Various regulatory agencies approved different methods- standard
and recommended method
• Some of the publications in the United States that are approved by
the regulatory agencies are Standard Methods for the Examination of Dairy
Products, Standard Methods for the Examination of Water and Waste Water, Standard
Methods for the Examination of Seawater and Shellfish, Compendium of Methods for the
Microbiological Examination of Food (all four are published by the American Public Health
Association, Washington, D.C.)
• Bacteriological Analytical Manual of Food and Drug Administration (prepared by the FDA
and published by the Association of Official Analytical Chemists, Arlington, VA)

5. Sampling for microbial analysis
• Sample
• Microbiological quality of a batch or a lot of a food
• Type of food and sample size
• True representative
• Sampling plans
• Single attribute plan: Sample is collected once and tested for bacteria
of interest. The results are used for acceptance or rejection of batch,
where n is sample size or number of sample, which dependents on
the batch or lot size (N). Defective unit(s) (C) of n is dependent on the
microbial type

6. • Multiple attributes plan: Initially based on the N the number of
samples (n1) are tested. If the C1 is above the standard level, a second
sampling is taken (n2) analyzed for C2 for decision
• Three- class attribute plan: Microbial levels are divided into three
groups: ≤ m is acceptable;
• > m but ≤ M is marginal;
• and > M is unacceptable. Number of units (C1) allowed between > m
and ≤ M among a number of samples tested (n) is defined for a
microbial type

7. • Plan for low level of contamination: This is important for some highly
potent pathogens.
• Units, depending upon the lot size, are tested to determine the
presence or absence of a pathogen in the unit to make a decision for
acceptance or rejection of a lot.

8. Sampling Procedure
Aspetical transfer
by following
sanitary measures
Storage condition
Sample
information
Media

9. Quantitative methods for Microbial
Enumeration in Foods
• Direct Enumeration
• Microscopic counts: Stained cells under bright field or live cells under
a phase contrast microbes can be enumerated using appropriate
microscopic factor, these counts- per gm or ml.
• Viable and non-viable cells
• Direct Epifluorescent filter technique (DEFT) is prefer for viable and
non viable
• Uses Acridine orange dye

10. • Liquid food
• Filter through membrane
• Acridine orange: fluorescent dye pour through filter
• Epifluorescent microscopy
• Count: manual or automatic
• Acridine orange binds to:
• RNA --- orange fluorescence
• DNA --- green fluorescence

11. Yeast cells by DEFT

12. Colony-forming units (CFU) in non-selective
agar media
Aliquots form selected dilutions of a serially
diluted sample
Bulk seeding or Surface spreading
Media- GYEA, tryptic soy agar, nutrient agar,
brain-heart infusion agar, and others.
For various groups Aerobic mesophile,
anaerobes, thermoduric, thermophile,
psychrophile, psychrotrophic & facultative
anaerobes

13. CFU in Nonselective Differential Chromogenic
media
Non-selective
medium is
supplemented
with an agent
capable of
differentiating
the colonies
produce by
organisms
Difference in
metabolic
pathway
Color change
due to pigment
production,
enzymes &
products
affecting pH of
medium
Use of pH
indicator and
substrate
Proteolytic,
lipolytic &
pectinolytic
microbial
groups in a
food

14. CFU in selective Agar media
• Selective or inhibitory agent
• Specific groups of microbes halophile osmophile & aciduric
Cetrimide agar Bromocresol purple lactose agar Tryptose sulfite cyclosporine agar

15. CFU in Selective-Differential Chromogenic
Agar Media
Media supplemented with
one or more selective
agents to allow selective
growth of specific resistant
microbes
Example; MacConkey agar,
Baired-Parker agar or V-J
agar, KF azide agar, & XLD
agar

17. Listeria monocytogenes on PALCAM
Listeria monocytogenes on MOX agar

18. Indirect Estimation
Dilution to
Extinction in
Nonselective broth
Most Probable
Number (MPN) in
selective Broth
Dye Reduction Test

19. Dilution to Extinction in Nonselective Broths
Sample is serial
diluted
Non selective
broth tryptic soy
broth
Determination
of growth in the
form of turbidity
Not a preferred
method in food
analysis

20. Most Probable Number (MPN) in Selective
broth
Sample is diluted
Inoculated in 3 or
5 broth of
selective medium
May or may not
contain inverted
durham’s tube
Each dilution is
scored for
presence or
absence of growth
Automated MPN
analysis system
(TEMPO)

21. Dye Reduction Test

24. Enumeration of Injured Microbial Groups by
Selective Media
Sub-lethally
injured microbes
Allow to repair the
in non-selective
media broth &
agar
Regain its ability
Than sub-cultured
in selective
medium
To ensure growth
of all type of
microbes
Standard and
recommended
method for plating

25. Qualitative Methods to Isolate Micro-
organisms in Foods
Isolation of pathogens
• Sub-lethally injured microbes
• Allow to repair the in non-selective media broth & agar
• Regain its ability
• Than sub-cultured in selective medium
• Alkaline peptone water, E.C broth, 7% NaCl N.B, Gram
Negative Broth & Rapport Vassiladis broth.

26. Test for Bacterial Toxins in Foods
• Bacteria and fungi such as Vibrio parahemolyticus, Staphylococcus
aureus, Clostridium botulinum, Bacillus cereus & molds produces
toxins
• In order to determine toxin concentration of toxin
• Detection is done by microslide precipitation method
• Radioimmunoassay (RIA), ELISA, & reverse passive latex agglutination
assay.
• Animal models

27. Rapid methods and automation
• Rapid
• Specific
• Sensitive and relatively accurate
• Less labor intensive

28. Metabolic Fingerprinting
• Each species of bacterium, mold, or yeast has unique C or N re